Neurobiology of Disease 146 (2020) 105139

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Neurobiology of Disease

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Review The in stem biology and neural development T ⁎ Caroline Vissersa,b, Aniketa Sinhab, Guo-li Mingc,d,e,f, Hongjun Songc,d,e,g, a Biochemistry, Cellular and Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA b Department of Biochemistry and Biophysics, Department of Psychiatry, University of California at San Francisco, San Francisco, CA 94158, USA c Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA d Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA e Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA f Department of Psychiatry, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA g The Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

ARTICLE INFO ABSTRACT

Keywords: The blossoming field of epitranscriptomics has recently garnered attention across many fields by findings that Epitranscriptome chemical modifications on RNA have immense biological consequences. of inRNA, m6A 6 6 1 including N6-methyladenosine (m A), 2-O-dimethyladenosine (m Am), N1-methyladenosine (m A), 5-methyl- Stem cells cytosine (m5C), and isomerization of uracil to (Ψ), have the potential to alter RNA processing Brain development events and contribute to developmental processes and different diseases. Though the abundance and roles of Brain disorders some RNA modifications remain contentious, the epitranscriptome is thought to be especially relevant instem cell biology and neurobiology. In particular, m6A occurs at the highest levels in the brain and plays major roles in embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. However, studies in these areas have reported conflicting results on epitranscriptomic regulation of stem cell pluripotency and mechanisms in neural development. In this review we provide an overview of the current understanding of several RNA modifications and disentangle the various findings on epitranscriptomic regulation of stemcell biology and neural development.

1. Introduction DNA and (Wang and Yi 2019). In fact, over 100 “epitran- scriptomic” modifications have been identified, though their biological The central dogma of biology—that information flows from DNA to consequences are largely unknown (Cantara et al. 2011; Machnicka RNA to protein—has acquired an increasing number of caveats and fine et al. 2013). A particular methylation at the N6 position of , print (He 2019). Beyond direct exceptions to the rule, like non-coding termed m6A, has garnered the most attention for its powerful role in , the details of the process are marred by questions like how much regulating mRNA processing (Roundtree et al. 2017). RNA is made from DNA, and how is this RNA processed to make the While the existence of m6A has been known for almost 50 years correct quantities of particular or isomers at the correct time? (Desrosiers et al. 1974; Lavi and Shatkin 1975; Rottman et al. 1974; Wei How might the system change in response to stimuli, and what reg- et al. 1975; Wei and Moss 1977), a combination of events reinvigorated ulatory systems drive these dynamics? The first step of the process, DNA a recent interest in the field. First, Zhong et al. showed in 2008 that to RNA, unfolds into many fields, including chromatin regulation, m6A is critical for developmental processes in Arabidopsis thaliana, epigenetics, and transcription. For example, reversible chemical mod- which suggested its importance for multicellular , and pu- ifications are dynamically added to DNA and histones to altergene shed for the development of m6A mapping methods (Zhong et al. 2008). expression and RNA levels (Akichika et al. 2019; Guo et al. 2011; Kohli Next, the discovery of the m6A demethylases, FTO and ALKBH5, in and Zhang 2013; Strahl and Allis 2000). The next step, making protein 2011 and 2013, respectively (Jia et al. 2011; Zheng et al. 2013), sug- from RNA, has largely been focused on the regulation of . gested that the m6A system may be dynamic, which implies that it can However, the recent exploration of post-transcriptional modifications be used as a regulatory system to alter mRNA fate in a context-depen- on RNA has shown that RNA is much more than a middleman between dent manner. Concurrently, antibody-based m6A mapping techniques

⁎ Corresponding author at: Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail address: [email protected] (H. Song). https://doi.org/10.1016/j.nbd.2020.105139 Received 7 April 2020; Received in revised form 9 October 2020; Accepted 11 October 2020 Available online 13 October 2020 0969-9961/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139 were developed and showed that m6A additions to mRNA are non- . However, the in vivo activity of FTO as an m6A demethylase random, thus pushing biological interest forward and providing tech- has recently been questioned, with the suggestion that it may instead 6 6 niques to understand the molecular mechanisms of m A in the cell act on m Am (Engel et al. 2018; Koh et al. 2019; Linder et al. 2015; (Dominissini et al. 2013; Meyer et al. 2012). In this review, we first Mauer et al. 2017; Schwartz et al. 2014b). On the other hand, one re- 6 6 1 introduce several epitranscriptomic marks that have garnered the most cent study reported that FTO can demethylate m A, m Am, and m A attention over recent years, with a focus on m6A. We then discuss the depending on the nuclear or cytoplasmic localization (Wei et al. 2018). major advancements in our understanding of the epitranscriptome in Yet another study argued that since loss of FTO has little effect on cy- 6 6 stem cell biology, especially embryonic stem cells (ESCs) and induced toplasmic mRNA m A or m Am levels, FTO likely functions primarily in pluripotent stem cells (iPSCs). We also touch on how the epitran- the nucleus. They further showed that methylation on small nuclear scriptome itself is regulated in embryonic stem cells. In addition, we RNAs (snRNAs) is a substrate for FTO demethylation (Mauer et al. review the role of the epitranscriptome in cortical and cerebellar de- 2019). The specificity of FTO remains a major hurdle in the fieldof velopment, as well as in adult neurogenesis. Finally, we discuss the role m6A; confirming its target is critically important so as not tomis- of the epitranscriptome in neurological disorders. attribute a phenotype or biological function to the wrong epitran- scriptomic mark. Finally, full knockouts of either ALKBH5 or FTO are 2. RNA modifications of particular interest not lethal in mice, though they appear to be particularly important in the cellular stress responses (Cao, 2019; Engel et al. 2018; Ma et al. 2.1. N6-methyladenosine: m6A 2018). The highly variable functions of m6A can be attributed to its many On a global level, 0.2 to 0.5% of all adenines are m6A modified distinct reader proteins. The central group of readers is the YTH-do- (Geula et al. 2015). The highest levels occur in the brain, where up to main-containing family of proteins, which bind directly to m6A. These 30% of all transcripts are modified (Chang et al. 2017). Epitran- readers have recently been reviewed elsewhere (Patil et al. 2018; scriptomic detection technologies have been focused on m6A, making it Roundtree et al. 2017). Briefly, YTHDC1 is found in the nucleus and one of the best-studied modifications to date.6 m A occurs in various regulates splicing, while YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are types of RNA, including tRNA, rRNA, non-coding RNA (ncRNA), and cytoplasmic and are thought to have distinct functions. YTHDF1 pro- mRNA. In 2012, two groups independently reported anti-m6A antibody- motes translation, YTHDF2 promotes mRNA degradation, and YTHDF3 based m6A RIP-Seq (MeRIP-Seq) techniques (Dominissini et al. 2012; seems to promote either translation or degradation in a context-specific Meyer et al. 2012). Subsequent mapping of m6A in the manner. Finally, the binding specificity and function of YTHDC2 re- showed that it is most commonly added at a consensus sequence of main unclear and may only be functional under special cellular con- DRACH (D = A, U or G; R = G or C; H = A, U, or C). m6A is especially ditions (Patil et al. 2018). In contrast to the notion that each YTHDF enriched in the 3’UTR and around the (Dominissini et al. reader promotes a unique fate of m6A-modified mRNA, two recent 2012; Meyer et al. 2012). While m6A does not alter Watson-Crick- studies found that YTHDF-1, 2, and 3 are jointly promote mRNA de- Franklin base pairing, it can modify protein binding and affect the gradation in cell lines (Zaccara and Jaffrey 2020) (BioRxiv: doi: https:// mRNA secondary structure (Farre et al. 2003; Roost et al. 2015). Many doi.org/10.1101/2020.06.03.131441). These divergent conclusions on m6A-binding proteins, or readers, have been identified. Individual the function of m6A reader proteins underscore the vital need for ad- readers confer unique downstream fates on m6A-modified mRNA, in- ditional research into reader protein function and regulation. cluding altered mRNA stability, translation, localization, and splicing. 6 6 m A methylation patterns in the transcriptome appear to be cell/tissue- 2.2. N6,2-O-dimethyladenosine: m Am specific and species-specific (Liu, 2019). Current m6A sequencing technologies are not sufficiently sensitive to profile6 m A at the the Unlike the internal m6A modification, N6,2’-O-dimethyladenosine 6 single-cell level, which would help quell disagreements in the field on (m Am) occurs in the mRNA terminus at the first following whether m6A is truly different across cell types or dynamic in response the N7-methylguanosine (m7G) cap. Approximately 0.0036% to 6 6 to stimuli. For example, Garcia-Campos et al. claimed that m A is “hard 0.0169% of all adenines are m Am modified when averaged across coded” and largely predictable in the yeast based on the ex- multiple human tissue types, corresponding to 526 to 1028 unique tended sequence around the modified site (Garcia-Campos et al. 2019). transcripts, depending on the tissue type (Liu, 2019). The number of 6 In contrast, studies that have profiled multiple mammalian tissues m Am-modified transcripts was previously thought to be much higher, 6 identified tissue-specific methylation profiles and significant changes but improved detection sensitivity has led to the view that m Am is only over development (Liu, 2019; Shi et al. 2018; Weng et al. 2018; Yoon moderately abundant (Frye et al. 2016). Three independent studies et al. 2017; Zhang et al. 2020). Furthermore, up-regulation of the m6A showed that phosphorylated C-terminal domain (CTD)-interacting 6 demethylases FTO and ALKBH5 in response to heat stress and hypoxic factor 1 (PCIF1) is a cap-specific m Am that targets stress, respectively, suggests that dynamic changes to the m6A methy- newly transcribed mRNA by associating with RNA Polymerase II lome may be a way of modulating cellular responses to stimuli (Zhang (Akichika et al. 2019; Boulias et al. 2019; Sun et al. 2019). By knocking 6 et al. 2016a; Zhang et al. 2016b; Zhou et al. 2015). Nonetheless, a lack out PCIF1 in various cell lines, Boulias et al. found that m Am most of reproducibility in m6A sequencing, especially using antibody-based strongly correlates with high expression and increased transcript sta- methods, may lead to false conclusions on the variability and dynamic bility (Boulias et al. 2019). However, this was not universally true for 6 6 6 nature of m A(McIntyre et al. 2020). all m Am-modified transcripts, leaving the regulatory capacity ofm Am 6 A growing list of proteins has been found to form the methyl- up for debate. The downstream functions of m Am are still unclear, with transferase complex that adds m6A onto mRNA (Fig. 1). This complex multiple conflicting studies reporting opposite effects on mRNA stabi- includes a core heterodimer unit of METTL3 and METTL14 (Liu et al. lity and translation (Akichika et al. 2019; Boulias et al. 2019; Liu, 2019; 2014), with accessory proteins including WTAP (Liu et al. 2014; Ping Sendinc et al. 2019). The field would greatly benefit from identification 6 et al. 2014), HAKAI, KIAA1429 (Schwartz et al. 2014b), and RBM15/B of m Am reader proteins that could help disentangle its potential (Patil et al. 2016). This complex has been reviewed elsewhere (Balacco downstream functions. and Soller 2019; Garcias Morales and Reyes 2020). There are two known m6A demethylases, ALKBH5 and FTO (Jia 2.3. N1-methyladenosine: m1A et al. 2011; Zheng et al. 2013). ALKBH5 co-localizes with nuclear speckles, indicating that both methylation and demethylation occur in The abundance of m1A in cells remains under debate. Some studies the nucleus. On the other hand, FTO can act in the nucleus and found that 0.015% to 0.16% of adenines are m1A modified,

2 C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139

Fig. 1. Overview of common epitranscriptomic marks. 6 7 1 6 5 This pinwheel shows the current knowledge for m A, m G, m A, Ψ, m Am, and m C (from top, clockwise). Each slice shows the known writer proteins, known eraser , known reader proteins and downstream functions of the epitranscriptomic mark. corresponding with over 4000 mRNA transcripts (about 20% of the promotes mRNA nuclear export through ALYREF, a nuclear m5C reader transcriptome) with high stoichiometries of m1A(Dominissini et al. protein (Yang et al. 2017). Additionally, m5C may cooperate with m6A 2016; Li et al. 2016). Two independent studies claimed that as few as 15 to enhance translation of particular transcripts like p21 (Li et al. or 53 total m1A sites exist in mRNA and lnRNA, and that it mostly 2017b). Finally, m5C addition to a subset of ncRNAs called vault RNAs occurs at low stoichiometries at these sites (Safra et al. 2017; Schwartz (vtRNAs) reduces downstream miRNA production (Hussain et al. 2013). 2018). In support of this, antibody cross-reactivity with m7G has been For example, NSUN2 regulates the processing of vault RNA, VTRNA1.1, shown to produce false positives in m1A identification, and a more into small-vault RNAs (svRNAs) that function similarly to miRNAs recent study found only one high-confidence, high stoichiometry m1A (Sajini et al. 2019). Though no m5C direct demethylases have been site using a bioinformatic approach, which was validated with an im- identified, ten-eleven translocation (Tet) enzymes can oxidize m5C to 5- proved m1A antibody (Grozhik et al. 2019). Though our understanding hydroxymethylcytosine (hm5C) and then unmodified cytosine (Ito et al. of the modification is limited, progress was made through identification 2011). The frequency of hm5C is about one hm5C per 5000 m5C(Fu of putative m1A (Fig. 1), namely, TRMT6/TRMT61A et al. 2014). This is slightly enriched in mRNA, with hm5C occurring on complex in the cytosol and TRMT10C/TRMT61B complex in the mi- ~7 × 10−6 of the total cytosines (Xu et al. 2016). In Drosophila, hm5C tochondria (Li et al. 2017c). Currently, ALKBH3 is the only known m1A was shown to preferentially mark mRNAs in coding regions and pro- mRNA demethylase, though it also acts on DNA and m3C in RNA (Aas mote their translation (Delatte et al. 2016; Fu et al. 2014). YBX1 is a et al. 2003; E et al., 2016; Ougland et al. 2004). m1A primarily exists in recently identified m5C reader protein that promotes stabilization of the 5’UTR near the translation initiation site, and its positive charge can modified mRNAs in early zebrafish embryos (Yang et al. 2019). One induce changes in secondary mRNA structure that may promote study showed that m5C can impair RBP binding, as is the case for translation (Dominissini et al. 2016; Li et al. 2017c). No studies of m1A SRSF2, which binds to unmethylated vtRNAs with higher affinity than in the brain have been reported, leaving a major gap in knowledge that to methylated RNAs (Sajini et al. 2019). will undoubtedly be explored in the coming years. 2.5. Pseudouridine: Ψ 2.4. 5-methylcytosine: m5C While pseudouridine (Ψ) is one of the most abundant modifications m5C is added to tRNA, rRNA, and mRNA by a variety of methyl- in ncRNA, its existence in mRNA is a recent finding (Carlile et al. 2014; transferases with specific RNA targets (Motorin et al. 2010). The re- Schwartz et al. 2014a). PUS1 and PUS7 enzymes isomerize to ported abundance of m5C on mRNA is extremely variable across dif- pseudouridine (Lovejoy et al. 2014) in an mRNA structure-dependent ferent studies, with some reporting up to 10,000 m5C sites in a single manner (Carlile et al. 2019)(Fig. 1). Other PUS-family proteins add Ψ tissue or cell type (Amort et al. 2017; Squires et al. 2012; Yang et al. to other types of RNA. On the other hand, no direct readers or removal 2017), while others using more stringent detection methods showed enzymes have been identified, raising the possibility that Ψ is irrever- that zero to only a few hundred sites are modified in mammalian mRNA sible. Some downstream effects of Ψ include weakening interactions (Huang et al. 2019b; Legrand et al. 2017). DNMT2 and especially between mRNA and Pumilio family proteins (PUFs) (Vaidyanathan NSUN2 are the most well-characterized m5C methyltransferases that act et al. 2017) and stabilizing RNA structure by improved base stacking on both tRNA and mRNA (Khoddami et al. 2019; Li et al. 2017b; Yang and increased hydrogen bonding (Carlile et al. 2014; Davis 1995; et al. 2017)(Fig. 1). NSUN2-mediated m5C mRNA methylation Spenkuch et al. 2014). Ψ has also been hypothesized to promote

3 .Vses tal. et Vissers, C. Table 1 Compilation of RNA modifications mapped in various cell types.

Mod. Tissue or cell type Detection method # of modified sites # of modified transcripts Comments Reference (peaks)

m6A Mouse ESCs m6A RIP-seq 8645 3942 annotated 6667 and 6159 peaks in Mettl3 KD and Mettl14 KD cells. Wang et al., Nat Cell Biology 2015 m6A Mouse ESCs m6A RIP-seq 9754 5461 mRNAs, 117 ncRNAs Average 2 peaks per transcript, including on core pluripotency Batista et al., Cell Stem Cell 2015 factors m6A Mouse naïve ESCs m6A RIP-seq 10,431 6412 Geula et al., Science 2015 m6A Mouse embryonic m6A RIP-seq 16,487 – 10,609 peaks are WTAP-dependent (64.3%) Schwartz et al., Cell Reports fibroblasts 2017 m6A Total mouse brain m6A RIP-seq 13,471 4654 coding Meyer et al., Cell 2012 236 non-coding m6A Mouse cerebellum m6A RIP-seq with bioinformatic 18,594 10,483 mRNAs 16,576 peaks shared with cortex, and 8924 mRNAs shared with Chang et al., Open Biology 2017 6 correction for m Am cortex m6A Mouse cortex m6A RIP-seq with bioinformatic 18,032 9636 mRNAs 16,989 peaks shared with cerebellum, and 8924 mRNAs shared Chang et al., Open Biology 2017 6 correction for m Am with cerebellum m6A E13.5 mouse cortex m6A RIP-seq 4055 2179 mRNAs Yoon et al., Cell 2017 m6A PCW11 human cortex m6A RIP-seq 10,980 5960 mRNAs Yoon et al., Cell 2017 m6A Mouse P7 cerebellum m6A RIP-seq 19,459 10,449 total Increases to 20,722 peaks on 11,215 RNAs upon hypobaric Ma et al., Genome Biology 2018 9879 mRNA hypoxia exposure m6A/m Mouse cortex m6A RIP-seq 14,656 7982 25 m6A/m peaks (in 20 ) were significantly modified in Engel et al., Neuron 2018 response to 4 h stress m6A Human brainstem m6A RIP-seq 30,459 12,659 Donor ID 5 Liu et al., Mol Cell 2019 m6A Human cerebellum m6A RIP-seq 45,975 14,048 Donor ID 5 Liu et al., Mol Cell 2019 m6A Human cerebrum m6A RIP-seq 28,001 11,770 Donor ID 5 Liu et al., Mol Cell 2019 m6A Human hypothalamus m6A RIP-seq 26,993 11,751 Donor ID 5 Liu et al., Mol Cell 2019 6

4 m A HEK293 CITS miCLIP, calling 9536 – 12,051 residues identified by truncation calling, and 33,157 Linder et al., Nature Methods identified by RIP-seq 2016 m6A HEK293T m6A RIP-seq 18,756 5768 43% conserved transcripts with mouse brain Meyer et al., Cell 2012 m6A HeLa cells m6A-CLIP 37,557 – ~90% peaks are deposited at pre-mRNA stage, 99% are constant Ke et al., Genes Dev 2017 between nucleus and cytoplasm 6 m Am HEK293 CITS miCLIP 487 – Linder et al., Nature Methods 2016 6 m Am HEK293 miCLIP 2350 – Compared miCLIP in PCIF1 KO cells and WT cells to distinguish Boulias et al., Molecular Cell 6 6 m Am and m A 2019 6 6 m Am MEL624 m Am-Exo-Seq – 1521 Compared PCIF1 WT and KO cells Sendinc et al., Molecular Cell 2019 6 6 m Am Human brainstem m A RIP-seq peaks at TSS 511 497 annotated Donor ID 5; one peak per transcript Liu et al., Mol Cell 2019 6 6 m Am Human cerebellum m A RIP-seq peaks at TSS 601 583 annotated Donor ID 5; one peak per transcript Liu et al., Mol Cell 2019 6 6 m Am Human cerebrum m A RIP-seq peaks at TSS 513 494 annotated Donor ID 5; one peak per transcript Liu et al., Mol Cell 2019 6 6 m Am Human hypothalamus m A RIP-seq peaks at TSS 472 455 annotated Donor ID 5; one peak per transcript Liu et al., Mol Cell 2019 m1A HeLa m1A RIP-seq 7154 4151 coding Average of 1.4 peaks per methylated Dominissini et al., Nature 2016 63 non-coding m1A HEK293T m1A RIP-seq 2129 – Dominissini et al., Nature 2016 Neurobiology ofDisease146(2020)105139 m1A HEK293T m1A-ID-seq 901 841 mRNAs 1989 peaks identified in ALKBH3 KO cells Li et al., Nature Chemical Biology 46 ncRNAs 2016 m1A HEK293T m1A RIP-seq 277 226 tRNA Safra et al., Nature 2017 28 mitochondrial 8 rRNA 15 mRNA/other m1A HEK293T m1A-MAP 740 473 mRNA and lncRNAs Subset of 53 sites is strongly enriched fro GUUCRA motif Li et al., Molecular Cell 2018 m1A HeLa RBS-Seq 91 88 tRNA mRNA peaks identified only if less stringent filters used Khoddami et al., PNAS 2019 2 rRNA 1 snoRNA m5C HeLa RNA-BisSeq 5399 1995 mRNAs 45% in CDS Yang et al., Cell Research 2017 336 ncRNAs (continued on next page) .Vses tal. et Vissers, C. Table 1 (continued)

Mod. Tissue or cell type Detection method # of modified sites # of modified transcripts Comments Reference (peaks)

m5C Mouse small intestine RNA-BisSeq 12,201 3722 annotated Peaks counted that are modified in at least 10% ofm1A sites in Yang et al., Cell Research 2017 rep 1 m5C Mouse heart RNA-BisSeq 12,397 3915 annotated “ Yang et al., Cell Research 2017 m5C Mouse muscle RNA-BisSeq 11,721 3624 annotated “ Yang et al., Cell Research 2017 m5C Mouse brain RNA-BisSeq 12,032 3635 annotated “ Yang et al., Cell Research 2017 m5C Mouse kidney RNA-BisSeq 12,149 3800 annotated “ Yang et al., Cell Research 2017 m5C Mouse liver RNA-BisSeq 11,414 3751 “ Yang et al., Cell Research 2017 m5C Mouse testis 3 W RNA-BisSeq 11,853 2727 “ Yang et al., Cell Research 2017 m5C HeLa RBS-Seq 487 340 annotated High-threshold cutoff Khoddami et al., PNAS 2019 5hmC Drosophila S2 cells hMeRIP-seq 3058 1597 coding Confirmed via dTet KD Delatte et al. Science 2016 Ψ HeLa RBS-Seq 754 322 mRNA HeLa cells collected with rRNA depleted total RNA, PolyA Khoddami et al., PNAS 2019 44 pseudogenes selected mRNAs & size selected small RNAs 55 rRNA 246 tRNA 24 snRNA 41 snoRNA 22 ncRNA Ψ HeLa Pseudo-seq 96 mRNA 88 mRNAs Carlile et al., Nature 2014 12 ncRNA 9ncRNA Ψ S. cerevisiae (budding) Pseudo-seq 260 mRNA 238 mRNA Lower threshold filter shows 466 Ψ on mRNAs. Carlile et al., Nature 2014 74 ncRNA 40 ncRNA Ψ S. Cerevisiae (midlog) Ψ-seq 328 168 mRNA Change under heat shock Schwartz et al., Cell 2014 24 ncRNA 46 tRNA

5 Ψ HEK293 and fibroblasts Ψ-seq 396 322 mRNA All human sites were combined into one dataset Schwartz et al., Cell 2014 28 ncRNA m7G mESCs m7G MeRIP 184 184 tRNa Functionally confirmed in Mettl1 KO mESCs Lin et al., Mol Cell 2019 m7G HeLa and HepG2 m7G meRIP, 801 681 mRNA present in two replicates in both HeLa and HepG2 cells Zhang et al., Mol Cell 2019 m7G-seq

The number of modified sites (peaks) and total number of unique transcripts that contain at least one modification varies greatly by tissue or cell type, detection method,andlab. Neurobiology ofDisease146(2020)105139 C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139 translation efficiency, though this remains to be proven(Carlile et al. 3.2. m5C in mESCs 2014). While many modifications have been identified, accurately mapping While the overwhelming focus of research in mESCs has been cen- modifications has been difficult. Not only are detection strategies lar- tered around m6A, m5C has also been examined. In one study, 12,492 gely limited to antibody-based methods, but different labs mapping m5C sites were identified in nuclear mESC mRNA. Modified mRNAs modifications in the same cell or tissue type have obtained largely were enriched for gene ontologies corresponding to cell cycle, RNA variable results (Table 1). Ongoing efforts to improve reproducibility processing, chromatin modification, and developmental processes. will be crucial for understanding the biological function of each mod- Though the functionality of m5C in mESCs was not shown, a correlation ification. between m5C sites and RBP sites was identified (Fig. 2). Approximately 29% of m5C sites in mESCs overlap with known RBP sites. More spe- 3. Epitranscriptomics in stem cell biology cifically, the largest overlaps correspond to UPF1 binding, which reg- ulates nonsense-mediated RNA decay. Additionally, SRSF3 and SRSF3 Epitranscriptomics appears to be especially important in stem cell splicing factors and the PRC2 subunit EZH2 have binding sites that biology, as it contributes to self-renewal and differentiation capacity. significantly overlap with m5C sites. This led to the hypothesis that m5C m6A is by far the most studied RNA modification in stem cells, parti- may contribute to RBP binding and functionality, though this requires cularly in ESCs and iPSCs. further validation (Amort et al. 2017). Finally, a recent study showed that during the maternal-to-zygotic transition in zebrafish develop- 3.1. m6A in ESCs ment, the m5C reader, YBX1, binds to m5C-modified mRNA to promote stabilization of maternal mRNAs during the transition (Yang et al. Early reports of m6A in ESCs were somewhat conflicting. One study 2019). reported that knockdown of Mettl3 and Mettl14 reduces m6A abundance As detection of diverse mRNA modifications continues to improve and impairs stem cell self-renewal (Wang et al. 2014b), whereas an- and orphan methyltransferase targets are identified, we expect our other study reported that Mettl3 knockout in mESCs improves self-re- understanding of epitranscriptomic regulation of stem cells to grow newal but blocks differentiation (Batista et al. 2014). However, both of rapidly. Notably, the low stoichiometry of some modifications relative these studies examined mESCs in vitro, which muddies our under- to m6A should not decrease their perceived importance, as the power of standing of the exact stage the ESCs are in and what m6A might do to the modification is derived from the strength of its downstream effects, drive embryonic development in vivo. This gap was addressed by Geula which vary widely among reader proteins. et al., who examined m6A in naïve pluripotent mouse ESCs. Naïve mESCs exist in a distinct molecular state compared to more advanced, 3.3. Induced pluripotent stem cells (iPSCs) “primed” epiblast stem cells (EpiSC). By knocking out Mettl3, they identified6 m A as a key driver of termination of the naïve state and The understanding that m6A contributes to pluripotency and dif- entry into the primed state, which is necessary for proper lineage dif- ferentiation drove studies of its regulatory capacity in iPSCs. In 2015, ferentiation at the post-implantation embryonic stage. The effects of Chen et al. showed that high abundance of m6A increases the repro- impaired differentiation are so drastic that loss ofm6A causes early gramming efficiency of mouse embryonic fibroblasts (MEFs) to plur- embryonic lethality (Geula et al. 2015). Importantly, this study further ipotent stem cells, in part by altering expression of key pluripotency clarified that6 m A regulates the genes governing both naïve and primed factors like Oct4, Sox2, and Nanog. This study further found an interplay states, and that loss of m6A causes upregulation of whichever genes are between microRNA (miRNA) binding to mRNA and enhanced Mettl3 modified in that particular stem cell state. Naïve mESCs show enhanced binding to mRNA to promote de novo addition of m6A(Chen et al. pluripotency upon Mettl3 knockdown, whereas primed EpiSCs show 2015). This concept of m6A interplay with noncoding RNAs has been increased stability of lineage-commitment genes upon loss of m6A explored with contrasting conclusions, and has been reviewed in-depth (Geula et al. 2015; Zhao and He 2015). Mechanistically, this study and elsewhere (Fazi and Fatica 2019). Furthermore, Chen et al. found that others determined that m6A primarily functions in development by Mettl3 knockdown reduces iPSC colony formation (Chen et al. 2015). reducing mRNA stability, which allows for the clearance of key naïve However, Geula et al. found that Mettl3 knockdown does not impair pluripotency-promoting transcripts or pro-differentiation transcripts, reprogramming efficiency, but rather slows proliferation of iPSCsin depending on the stem cell stage (Fig. 2)(Batista et al. 2014; Geula early reprogramming (Geula et al. 2015). Finally, Wu et al. found that et al. 2015; Wang et al. 2014a; Wang et al. 2014b). Mettl3 knockdown decreases the proliferation rate of porcine iPSCs In addition to the traditional METTL3/METTL14-mediated addition (piPSCs) and impairs expression of key pluripotency genes, though they of m6A to mRNA, there are several other m6A methyltransferases. In did not test for reprogramming efficiency. This study further identified particular, METTL16 was identified in human cells as anm6A methyl- that m6A promotes YTHDF1-mediated translation of JAK2 in piPSCs, transferase that primarily targets small nuclear RNA (snRNA), specifi- while promoting degradation of SOCS3 via YTHDF2 (Wu et al. 2019). cally U6 snRNA, and other non-coding RNAs (Warda et al. 2017). Ad- Both of these mechanisms lead to upregulation of the JAK2-STAT3 ditionally, METTL16 regulates expression of the SAM synthetase signaling pathway, which is known to promote stem cell self-renewal by MAT2A (Pendleton et al. 2017), which is highly consequential for all increasing expression of the core pluripotency genes Klf4 and Sox2 modifications that use SAM as a methyl donor (Fig. 2). Accordingly, (Niwa et al. 1998; Wu et al. 2019). In parallel, YTHDF2 has been shown Mendel et al., 2018 found that METTL16-mediated modification of to be upregulated in iPSCs to destabilize m6A-modified mRNAs related Mat2a mRNA is necessary for proper embryonic development of mouse to neural development and thereby promote pluripotency (Heck et al. blastocysts, and homozygous knockout of Mettl16 is embryonic lethal. 2020). Analysis of E2.5 Mettl16 KO mouse blastocysts showed that only 20 Overall, m6A clearly regulates the pluripotency of iPSCs, but its role genes are differentially expressed relative to the wildtype, with Mat2a in reprogramming likely depends on the cellular context of the starting showing the most significant downregulation. However, by E3.5 the material or the stage of reprogramming. As was the case in ESCs, m6A global transcriptome was massively dysregulated (Mendel et al. 2018). may alter expression of the gene transcripts already present. Still, fur- While the more common METTL3/METTL14-mediated pathway has ther investigation is needed to identify the fate of m6A-modified tran- garnered the most attention, understanding the complexities of the scripts. While m6A-mediated mRNA degradation appears to be a major epitranscriptome and the consequences of mediating highly con- mechanism, expression of other reader proteins suggests a more com- sequential genes like Mat2a will be necessary to accurately characterize plex system. Understanding how m6A reader proteins selectively bind the many roles of m6A. particular mRNA targets will be a major step forward in further

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Fig. 2. The epitranscriptome in embryonic development and ESCs. Top: Marks that have been studied in the developing embryo include m6A on mRNA to promote mRNA degradation, m7G on tRNA to promote translation, METTL16- mediated addition of m6A to ncRNA like Mat2a, m5C on mRNA to promote RNA Binding Protein (RBP) mRNA binding, and m5C on mitochondrial tRNA to regulate germ layer specification. Bottom: The most-studied modification in embryonic stem cells (ESCs)ism6A. While several conflicting studies have reported different phenotypes after Mettl3 knockdown, the current consensus is that m6A functions to destabilize whichever gene transcripts are modified at the time. In naïve ESCs purified from pre-implantation blastocysts, m6A is primarily added to pluripotency-promoting gen transcripts. Therefore loss of m6A improves self-renewal and impairs differentiation. In contrast, in primed ESCs derived from post-implantation blastocysts,6 m A is primarily added to lineage-commitment gene transcripts, so loss of m6A promotes differentiation and impairs self-renewal. elucidating the mechanisms of m6A action in iPSCs. 2018; Wen et al. 2018). Zc3h13 can form a complex with WTAP, Virilizer (Kiaa1429), and Hakai, which also contribute to the METTL3- METTL14 m6A methylation complex (Horiuchi et al. 2013; Wan et al. 3.4. Regulation of stem cell epitranscriptomes 2015). Wen et al. then showed that Zc3h13 knockdown in mESCs de- 6 6 creases global m A levels to about 30–40% as in the control, and con- Upstream regulation of m A deposition or differential expression of 6 6 firmed m A dependency on Zc3h13 through MeRIP-seq. More specifi- the writers, readers, and erasers contributes to the function of m A in cally, Zc3h13 promotes m6A deposition by localizing the Zc3h13- stem cells. For example, Aguilo et al. showed that zinc finger protein 6 WTAP-Virilizer-Hakai complex to nuclear speckles; loss of Zc3h13 217 (ZFP217) coordinates epigenetic regulation with m A deposition. causes these complex components, as well as METTL3/METTL14, to More specifically, ZFP217 is a transcription factor that directly activates 6 significantly shift to localization in the cytoplasm. Functionally, Zc3h13 transcription of several key pluripotency genes, then blocks m A knockdown impairs mESC self-renewal, decreases expression of plur- modification of these genes by sequestering METTL3 in mESCs and 6 ipotency genes, and increases expression of differentiation markers in iPSCs. ZFP217 knockdown causes global increases in m A levels, which correlation with differential m6A modifications of these gene transcripts correlates with a decreased half-life of Nanog, Sox2, c-Myc, and Klf4 (Wen et al. 2018). The conclusion that m6A promotes self-renewal is mRNA transcripts. This in turn impairs pluripotency and reprogram- consistent with previous studies (Wang et al. 2014b), and the con- ming (Aguilo et al. 2015). sequences on pluripotency correspond to studies performed under si- Wen et al. found that another zinc-finger protein, Zc3h13, is critical 6 milar conditions in mESCs (Geula et al. 2015). These two studies on for m A deposition, and Zc3h13 knockdown significantly impairs self- zinc finger proteins are important examples of6 howm A may be renewal and maintenance of pluripotency in mESCs (Knuckles et al.

7 C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139 regulated or targeted to individual transcripts in stem cells. This con- neuronal markers in wildtype NPCs allow for pre-patterning of differ- nection between transcription factors and epitranscriptomic regulation entiation by allowing transcription of pro-neuronal genes but pre- remains an interesting avenue for further research. venting significant protein production (Yoon et al. 2018). Finally, we Finally, one study has found a direct connection between histone used iPSC-derived human brain organoids to confirm that m6A also methylation and sites of m6A deposition. Huang et al. showed that regulates NPC cell cycle progression in humans (Yoon et al. 2017). We histone H3 trimethylation at lysine-36 (H3K36me3) drives m6A me- then compared m6A-seq analysis among human brain organoids, thylation by recruiting and promoting interactions between the m6A human post-conception week 11 embryonic brain tissue and E13.5 methyltransferase complex and its target mRNA. More specifically, mouse brains. While many gene transcripts are m6A-modified in both METTL14 binds to H3K36Me3, chromatin, and RNA, thereby pro- species, the human-specific modifications correlated strongly with dis- moting the co-transcriptional addition of m6A to genes with H3K36Me3 ease ontologies for human-specific mental disorders like autism and epigenetic marks. Knockdown of the H3K36Me3 methyltransferase, schizophrenia. This work provided the first in vivo analysis of m6A in SETD2, impairs binding of the m6A methyltransferase complex to sites mammalian brain development and highlighted the possibility that that lose H3K36Me3, and globally reduces m6A levels. In mESCs, m6A may contribute to psychiatric or neurodevelopmental disorders in SETD2 knockdown induces higher expression of pluripotency factors humans. (OCT4, SOX2, NANOG) and prevents increased m6A methylation during Shortly thereafter, an independent study by Wang et al. knocked out differentiation. This suggests that H3K36Me3 drives m6A modifications Mettl14 in the developing forebrain, and also found that loss of m6A to destabilize pluripotency genes and promote differentiation, and loss slows NPC cell cycle progression. In vitro analysis of Mettl14 cKO NPCs of either H3K36Me3 or METTL14 promotes pluripotency over differ- showed that loss of m6A can cause premature differentiation, and in vivo entiation (Huang et al. 2019a). This corresponds with previous reports analysis showed that Mettl14 cKO mice had reduced numbers of Pax6+ that m6A is necessary for proper differentiation of mESCs (Batista et al. NPCs and reduced numbers of Satb2+ late-born neurons. This led the 2014; Geula et al. 2015), and provides the first evidence that m6A ad- authors to suggest that depletion of the NPC pool causes a reduction in dition may be directed by epigenetic marks. neurogenesis (Wang et al. 2018b). This contrasted with our study, While a few studies have identified how the epitranscriptome may which showed an increase in Pax6+ cells in Mettl14 cKO forebrains, but be regulated, over 100 putative METTL3 or METTL14 binding proteins a similar decrease in late-born neurons; we therefore proposed that m6A have been identified, suggesting that there is much left to be learned is necessary for the timely differentiation of NPCs, and loss6 ofm A about upstream regulation of m6A(Malovannaya et al. 2011). A better causes a build-up of Pax6+ NPCs (Yoon et al. 2017). These differences understanding of how the methyltransferase complex and demethylases may stem from different methodologies or antibodies. Nonetheless, the target specific gene transcripts, as well as how writer, reader, and eraser studies agree that m6A regulates mRNA stability to alter gene expres- expression is regulated, will drive the field forward. sion and NPC fate. Next, Wang et al. identified genome-wide changes in histone mod- 4. Epitranscriptomics in neural development ifications upon Mettl14 knockout. Specifically, cKO NPCs show in- creases in histone H3 at lysine 27 (H3K27ac), histone H3 Recent work has shown that the epitranscriptome, in particular trimethylation at lysine 4 (H3K4me3), and histone H3 trimethylation at m6A, is especially important for neural development and brain function lysine 27 (H3K27me3). Chemically blocking these epigenetic marks (Livneh et al. 2020; Yoon et al. 2018). (Lence et al., 2016) performed partially rescues cKO NPC proliferation defects. The changes in histone one of the first studies of m6A in the brain, using Drosophila melano- modification were partially attributed tom6A-mediated destabilization gaster as a model organism. This study showed that m6A is enriched in of CBP and p300 transcripts, which are stabilized upon loss of m6A. the nervous system and that knockout of the methyltransferase com- However, this did not apply to transcripts in the PRC2 complex, sug- ponents causes reduced lifespan, severe behavioral defects, and global gesting there are also other mechanisms at play (Wang et al. 2018b). changes in neural (Lence et al. 2016). While this work Overall, the connection between the epitranscriptome and epigenetics was important for understanding m6A in vivo, it contrasted with in the developing brain is highly intriguing. As single-transcript m6A mammalian studies in that loss of m6A methyltransferases is not lethal editing techniques are developed (Wilson et al. 2020), it would be in flies. The next major advances came from studies of conditional pertinent to edit only CBP and p300 mRNA to quantify the degree to knockout of the m6A methyltransferase complex in mice to examine which their methylation contributes to the Mettl14 cKO phenotype, as epitranscriptomic regulation during mammalian brain development. opposed to the sum of many modified transcripts. Below we provide an in-depth overview of the epitranscriptome in Finally, a third study conditionally knocked out Ythdf2 in the de- mammalian neural development (Fig. 3). veloping forebrain to show that m6A largely functions through YTHDF2-mediated mRNA degradation during cortical development. In 4.1. Cortical development this study, Li et al. showed that Ythdf2 KO mice have a very similar phenotype to Mettl14 cKO mice. In particular, loss of Ythdf2 impairs In 2017, our laboratory showed that conditional knockdown (cKO) NPC proliferation and differentiation, and causes delays in cortical of Mettl14 in mice and subsequent loss of m6A in neural progenitor cells neurogenesis. They also found that Ythdf2−/− NPCs create fewer pri- (NPCs) drastically impairs brain development in vivo (Yoon et al. 2017). mary neurites per neuron and shorter neurites overall when differ- Loss of m6A impairs NPC differentiation, slows cell cycle progression, entiated in vitro, suggesting that m6A also regulates neuron maturation and elongates the timing of cortical neurogenesis into postnatal stages. during the differentiation process (Li et al. 2018b). This study was Mechanistically, m6A-modified genes are significantly enriched for necessary to confirm that6 m A regulation of cortical development gene ontologies that correlate with regulation of transcription, neuron functions primarily through YTHDF2-mediated mRNA degradation and differentiation, cell cycle, and stem cell differentiation. These modified that m6A promotes NPC proliferation and differentiation. transcripts have a shorter half-life than their corresponding unmodified Beyond m6A, m5C is also crucial for proper neurodevelopment. transcripts in Mettl14 cKO mouse NPCs, suggesting that m6A normally Flores et al. showed that the m5C methyltransferase NSUN2 is essential destabilizes mRNA in the developing brain. By modifying both multi- for neural stem cell differentiation (Flores et al. 2017). Loss-of-function potency and differentiation-promoting transcripts, the6 m A system may in Nsun2 caused neurodevelopmental defects such as mi- contribute to harmonious changes in gene expression that are necessary crocephaly in mouse and human models. In a conditional Nsun2 for the progression of NPCs through the distinct phases of embryonic knockout in the developing mouse brain, there were signs of decreased cortical neurogenesis. To this end, we found that Mettl14 cKO NPCs co- sizes of the cerebral cortex, hippocampus, and striatum compared to express stem cell and neuronal markers, and that rapid degradation of wildtype mouse brains. In addition, Nsun2 cKO mice had lower levels of

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Fig. 3. m6A in neural development. Top: In the cortex, loss of m6A elongates the timeframe of cortical neurogenesis such that the postnatal brain is still generating upper-layer neurons. This is accomplished through altered mRNA degradation rates of key pluripotency and fate-determining gene transcripts. Middle: In the cerebellum, loss of m6A causes disorganization of the Purkinje cell layer (PCL) into the inner granule cell layer (IGL). IGL cells also exhibit a higher rate of apoptosis, resulting in fewer total IGL cells. Mechanistically, m6A has been shown to promote and mRNA degradation in the cerebellum. Bottom: In hippocampal adult neurogenesis, in vitro studies found that loss of m6A impairs self-renewal and neurogenesis, but the mechanism of action remains unclear. global protein production and increased cellular stress compared to is turned ON at P14, P21, or P60 have gene ontologies enriched for wildtype brains. These phenotypes are thought to arise due to differ- signal transduction, cell adhesion, learning, and synaptic plasticity. entiation delays, lack of neural lineage commitment, and a reduction in Overall, m6A modification patterns strongly correlate with the pro- upper-layer neurons (Flores et al. 2017). This interesting study gression from proliferating cells at P7 to mature neuronal activities at broadens the importance of the epitranscriptome in neural development P60. This study also examined changes in expression of METTL3, beyond m6A. Indeed, as detection methods for other modifications METTL14, WTAP, FTO, and ALKBH5. Though cerebellar expression of become more reliable, it will be worthwhile to explore a greater di- all of these genes decreased on average over time, there was a specific versity of epitranscriptomic marks in neural development. reduction in internal granular layers but elevated expression in Purkinje cells. Lentiviral Mettl3 knockdown at P7 lowers the number of Purkinje cells and impairs their organization along the outer surface of the inner 4.2. Cerebellar development granule cell layer. On the other hand, Alkbh5-KO mice had no ob- servable phenotype in the cerebellum under normal conditions, which The complexity of the brain suggests that epitranscriptomic reg- may be due to redundant actions by FTO. After stressing the developing ulatory systems may have distinct functions in different parts of the brain with hypobaric hypoxia, Alkbh5-KO mice had significantly brain. Indeed, Chang et al. showed that m6A levels are increased in the smaller cerebella and fewer mature neurons, yet significantly more adult mouse cerebellum compared to the cerebral cortex, and that there proliferating cells. This suggests that ALKBH5 is critical for promoting are region-specific methylation patterns (Chang et al. 2017). Even cerebellar neurogenesis under stress. Finally, this study showed that within the cerebellum, methylation patterns change over development. several important gene transcripts are differentially localized in the Ma et al. showed that methylation targets change across postnatal day 7 cytoplasm over nucleus in Alkbh5-KO cerebella, indicating that m6A (P7), P14, P21, and P60 mouse cerebella. There are 12,452 m6A peaks promotes nuclear export in this tissue (Ma et al. 2018). that are turned “ON” (emerge at a later stage) over time, and 11,192 In contrast, Wang et al. used a Mettl3 cKO mouse model to show that that are turned “OFF” (disappear in later stages). The groups of tran- m6A promotes mRNA degradation and alternative splicing in the cer- scripts methylated at each time point correspond with the develop- ebellum. Mettl3 cKO mice have drastically smaller cerebella, sig- mental processes happening at that time. For example, gene transcripts nificantly fewer cerebellar granule cells (CGCs) in the internal granular in which m6A is turned OFF from P7 to P14 have gene ontologies en- layer (IGL), and disordered Purkinje cell organization relative to riched for cell cycle. On the other hand, gene transcripts in which m6A

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Fig. 4. The epitranscriptome in neural disorders. Top left: is correlated with m6A in that the central protein involved in Fragile X, FMRP, can bind m6A to promote nuclear export of modified mRNAs through interaction with CRM1, a component of the nuclear pore complex. Loss of FMRP in mice impairs this export and causes decreased levels of embryonic neurogenesis and NSC proliferation. Top right: PUS3 mutations in humans significantly correlate with intellectual disability and microcephaly. Though theexact mechanism is unknown, mutations in PUS3 cause significantly lower levels of pseudouridine addition on tRNA relative to wildtype controls. Bottomleft: PUS7 mutations identified in humans and validated in drosophila cause increased aggression, speech delay, intellectual disability, and microcephaly through decreased levels of pseudouridine on both tRNA and mRNA. Bottom right: METTL5 mutation in humans and validated in zebrafish and mice cause intellectual disability and microcephaly through decreased levels of m6A on 18S ribosomal RNA. wildtype controls. Furthermore, loss of m6A causes significantly in- 4.3. Adult neurogenesis creased levels of apoptosis of newborn granule cells, which explains the depletion of CGCs. Again, loss of m6A increases mRNA stability; m6A The m6A demethylase FTO has been implicated in numerous path- modifications on apoptosis-associated gene transcripts normally restrict ways in the mature brain, from (Cui et al. 2017), to psychiatric their expression. Notably, m6A-mediated regulation of apoptosis ap- and neurodegenerative diseases (Choudhry et al. 2013; Hess et al. 2013; pears to be specific to the cerebellum, as these transcripts are not sta- Keller et al. 2011; Li et al. 2018a; Widagdo et al. 2016), to regulation of bilized in the cortex of Mettl3 cKO mice. Finally, Wang et al. identified adult neural stem cells (Gao et al. 2010; Li et al. 2017a). However, an additional mechanism of m6A-mediated alternative splicing in the understanding the role of FTO remains difficult due to its multiple cerebellum. exclusion occurs more frequently upon m6A deple- functions in DNA and RNA demethylation. In fact, the first study on tion, especially in transcripts that are normally methylated in the FTO in neurogenesis was published in 2010, before FTO was even wildtype. These alternatively spliced transcripts are enriched for gene identified as an m6A demethylase (Gao et al. 2010; Jia et al. 2011). Gao ontologies in synapse-associated pathways and neurotransmitter re- et al. generated whole-body and neural-specific Fto KO mice and found ceptors. Further analysis showed that increases in intracellular calcium that the two have very similar phenotypes, indicating that the majority concentration in Mettl3 cKO CGCs contributes to their increased apop- of FTO functions occur in the nervous system (Gao et al. 2010). In 2017, tosis (Wang et al. 2018a). This work highlights the fact that epitran- it was shown that FTO is expressed in adult NSCs (aNSCs) and in mature scriptomic regulation is highly cell-type specific with unique roles in neurons and its expression increases over postnatal time. Fto KO mice different parts of the brain. How this specificity is regulated willbean show reduced proliferation and differentiation of aNSCs, which func- interesting avenue of future research. tionally impairs learning and memory. Furthermore, loss of FTO results

10 C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139 in slightly higher (~15%) levels of m6A, though only 363 genes are 5.2. Intellectual disability both m6A modified and differentially expressed upon loss of FTO (outof 5635 m6A-modified genes and 1862 FTO-dependent genes) (Li et al. Recent studies identified correlations between epitranscriptomic 2017a). While FTO does seem to regulate adult neurogenesis, the de- modifications and intellectual disability. First, Shaheen et al. found that gree to which this is enacted through m6A remains in question, espe- mutations in human PUS3, a pseudouridinylation , correlates cially considering that FTO can act on multiple targets in vivo. with intellectual disability and microcephaly in three affected siblings Next, Chen et al. found that Mettl3 knockdown impairs both pro- (Shaheen et al. 2016). The affected individuals also have a significant liferation and differentiation of aNSCs cultured in vitro. m6A sequencing reduction in Ψ-modified tRNA relative to healthy controls in purified showed that the m6A landscape is dynamic between proliferating and lymphoblastoid cells. The PUS3 deficiency phenotype in humans is differentiating cultured aNSCs; transcripts modified only inpro- largely brain-specific, suggesting that PUS3-mediated tRNA Ψ mod- liferating aNSCs correlate with cell cycle, while transcripts modified ification is especially important for cognitive function. only in differentiating aNSCs are enriched for protein localization, Next, both de Brouwer et al., 2018 and Shaheen et al., 2019 iden- signaling, and synapse organization (Chen et al. 2019). This study is tified mutations in PUS7, a tRNA and mRNA pseudouridinylation en- slightly more direct in studying m6A in adult neurogenesis by knocking zyme, that cause intellectual disability, microcephaly, speech delay, down Mettl3, but the use of cultured aNSCs limits the conclusions that and aggressive behavior (de Brouwer et al. 2018; Shaheen et al. 2019). can be drawn; aNSCs exist in highly specialized niches in vivo that are Ψ at position 13 in tRNA and PUS7 target mRNAs were significantly difficult to recapitulate in vitro (Ming and Song 2011; Song et al. 2012). reduced in affected individuals compared to healthy controls. Ad- Finally, a 2019 study found that Fto cKO in aNSCs decreases aNSC ditionally, Pus7 knockout in Drosophila recapitulates the cognitive im- proliferation and differentiation into NeuN+ neurons at 4 weeks after pairment phenotype and the molecular loss of Ψ at particular target FTO knockout. While the fate of m6A-modified transcripts was not sites (de Brouwer et al. 2018). This provides exciting evidence that Ψ tested, individual mRNA transcripts in the Stat3 signaling pathway, modifications of mRNA and tRNA are not only highly conserved across Socs5 and Pdgfrα, were shown to play important roles in FTO-mediated species, but are critical in neural development. Additional studies using regulation of aNSCs. However, Socs5 mRNA and protein decrease in Fto mouse models to investigate the exact mechanism of Ψ in neural de- cKO aNSCs, while Pdgfrα mRNA and protein increase (Cao, 2019). velopment will be an exciting next step. Therefore, the involvement of m6A and mechanisms of m6A-mediated A 2012 study by Abbasi-Moheb et al. showed that homozygous loss regulation in aNSC remain unclear. In multiple studies, effects of Fto or of the m5C methyltransferase, NSUN2, due to nonsense or splicing Mettl3 KD appear stronger in in vitro cultured cells than in vivo aNSCs. mutations in the transcript causes memory and learning deficits in The highly dynamic nature of m6A in response to signaling and stress humans (Abbasi-Moheb et al. 2012). Further experiments with a stimuli suggest that culturing systems need to be incredibly carefully knockout of the NSUN2 ortholog, CG6133, in Drosophila showed short- controlled to maintain an accurate representation of the epitran- term-memory deficits that could be rescued when the wild type protein scriptome in in vivo aNSCs. was expressed. The fact that humans with NSUN2 mutations show in- tellectual disabilities and facial dysmorphism, combined with the finding that similar phenotypes are found in Drosophila suggests that 5. Epitranscriptomics in neurodevelopmental diseases m5C may be a fundamental regulator of neural function across species (Abbasi-Moheb et al. 2012). These data strongly suggest that m5C In accordance with its powerful role in neural development, m6A writers are important in neural development and function, but further has been linked to neurodevelopmental defects as well. To date, m6A in studies are necessary to understand the mechanistic role of m5C in the Fragile X Syndrome is the best-characterized interaction. Additionally, brain. emerging genome-wide association studies and human genetics studies Finally, Richard et al. identified frameshift mutations in METTL5, have linked mutations in epitranscriptomic enzymes with intellectual which adds m6A to 18S rRNA (van Tran et al. 2019), that cause auto- disability (Fig. 4). somal-recessive intellectual disability and microcephaly. METTL5 is expressed in the human brain from early development and into adult- hood, particularly in the cerebellar cortex, hippocampus, and striatum. 5.1. Fragile X syndrome Analysis in rodents confirmed ubiquitous METTL5 expression in the brain, with increased staining in neural soma and nuclei, as well as in Fragile X mental retardation protein (FMRP), encoded by FMR1, is pre- and post-synaptic regions. Finally, Mettl5 knockout in zebrafish an RNA-binding protein that is best known for negatively regulating the recapitulates the microcephaly phenotype and specifically causes a translation of its target mRNAs (Darnell et al. 2011; Richter et al. 2015) decrease in forebrain and midbrain size (Richard et al. 2019). While and trafficking mRNA granules (De Diego Otero et al. 2002). Loss-of- mechanistic studies of METTL5 action have only recently begun, this function mutations in FMR1 cause Fragile X Syndrome, which is marked genetic evidence suggests that it is yet another epitranscriptomic by intellectual disability and delayed development. In 2017, Arguello modifier that is crucial for proper brain development. et al. identified FMRP as an m6A binding protein in vitro (Arguello et al. 2017). Zhang et al. then showed that FMRP binds to YTHDF2 and that 6. Concluding remarks and future outlook FRMP target genes are enriched for m6A marks in the mouse cerebral cortex (Zhang et al. 2018). A knockout of Fmr1 resulted in the down- The field of epitranscriptomics has reached a point where the power regulation of some m6A mRNA FMRP target transcripts, suggesting that of various mRNA modifications has become widely accepted, but the FMRP stabilizes these m6A modified mRNAs (Zhang et al. 2018). Next, specific mechanisms of their action remain under debate. It is becoming Edens et al. showed that FMRP promotes nuclear export of m6A-mod- increasingly important to perform extremely careful experiments to ified mRNA by interacting with CRM1, a nuclear export protein. Ad- detect and validate epitranscriptomic marks to prevent further confu- ditionally, Fmr1 KO mice phenocopy Mettl14 cKO mice in terms of sion regarding their downstream functions. Furthermore, expression of delayed embryonic cortical neurogenesis and prolonged NPC cell cycle multiple reader proteins and multiple published functions of m6A in a progression. In both of these mouse knockout models, FMRP target single cell type suggest that m6A may differentially regulate various mRNAs are retained in the nucleus (Edens et al. 2019). The binding gene transcripts within a single cell. Several important strategies to affinity of FRMP for6 m A-modified mRNA and its role in nuclear export further elucidate the regulatory capacities of m6A in stem cells and was recently confirmed by another study (Hsu, 2019). neural development include (1) improved detection techniques for higher sensitivity and accuracy, (2) studies on how reader proteins

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6 selectively bind a subset of m A-modified mRNAs, and (3) careful Desrosiers, R., et al., 1974. Identification of methylated in messenger RNA analyses and conservative interpretations of data to prevent over- from Novikoff hepatoma cells. Proc. Natl. Acad. Sci. U. S. A. 71, 3971–3975. Dominissini, D., et al., 2012. Topology of the human and mouse m6A RNA methylomes confident conclusions that will hinder future studies. revealed by m6A-seq. Nature. 485, 201–206. 6 In addition to clarifying studies on m A, we are particularly excited Dominissini, D., et al., 2013. Transcriptome-wide mapping of N(6)-methyladenosine by m by the prospects of other epitranscriptomics marks in neural develop- (6)A-seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 1 5 7 6 8, 176–189. ment and disease. Careful mapping of m A, m C, m G, m Am, and Ψ in Dominissini, D., et al., 2016. The dynamic N(1)-methyladenosine methylome in eu- the brain alongside generating animal knockouts of their respective karyotic messenger RNA. Nature. 530, 441–446. modifying enzymes will greatly expand the breadth of knowledge in the Edens, B.M., et al., 2019. FMRP modulates neural differentiation through m(6)A-depen- field of epitranscriptomics. With an increasing number of scientists dent mRNA nuclear export. Cell Rep. 28, 845–854 e5. Engel, M., et al., 2018. The role of m(6)a/m-RNA methylation in stress response reg- working in this field, we expect the next five years to be full ofnew ulation. Neuron. 99, 389–403 e9. discoveries with profound implications for basic and translational sci- Farre, D., et al., 2003. Identification of patterns in biological sequences at the ALGGEN ence. server: PROMO and MALGEN. Nucleic Acids Res. 31, 3651–3653. Fazi, F., Fatica, A., 2019. Interplay between N (6)-Methyladenosine (m(6)a) and non- coding RNAs in cell development and Cancer. Front. Cell Dev. Biol. 7, 116. Declaration of Competing Interest Flores, J.V., et al., 2017. Cytosine-5 RNA methylation regulates neural stem cell differ- entiation and motility. Stem Cell Reports. 8, 112–124. Frye, M., et al., 2016. RNA modifications: what have we learned and where arewe None. headed? Nat. Rev. Genet. 17, 365–372. Fu, L., et al., 2014. Tet-mediated formation of 5-hydroxymethylcytosine in RNA. J. Am. Chem. Soc. 136, 11582–11585. Acknowledgement Gao, X., et al., 2010. The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice. PLoS One 5, e14005. We thank K. M. Christian for comments. The work in the authors' Garcia-Campos, M.A., et al., 2019. Deciphering the “m(6)a code” via antibody-in- dependent quantitative profiling. Cell. 178, 731–747 e16. laboratories was supported by the National Institutes of Health Garcias Morales, D., Reyes, J.L., 2020. 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